Significance of the proposed research. Enteric bacterial pathogens cause food borne disease and thus are a source of enormous economic and health burdens. Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is especially important because it causes a severe bloody diarrhea following transmission to humans in contaminated beef and vegetable products. EHEC also causes a fatal kidney disease (hemolytic uremic syndrome;HUS) for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g. enteropathogenic E. coli (EPEC), Salmonella, Shigella, Yersinia) utilize a type III secretion system (T3SS) to inject virulence proteins (effectors) into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, the mechanisms of how effectors bind to host proteins and subsequently disrupt the normal function of intestinal epithelial cells are not characterized in sufficient detail to permit rational development of new therapeutic strategies to combat diarrheal disease. The central hypothesis for the proposed research is that the EHEC NleH1 and NleH2 effector proteins subvert the function of the host transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-:B) by binding to ribosomal protein S3 (RPS3). RPS3 is a transcriptional cofactor that provides specificity to NF-: B-dependent transcriptional outputs in response to various cellular stimuli. The specific aims of this project are to 1) Define the minimal NleH-RPS3 binding domains and the functional consequences of their binding, 2) Measure the influence of the kinase activities of NleH and cellular factors on RPS3 function in nuclear localization and transcription, 3) Quantify the extent to which NleH alters host transcriptional responses to bacterial infection, and 4) Measure the contribution of NleH to bacterial virulence in animal models of disease caused by attaching/effacing pathogens. This project is significant and innovative because it will test the hypothesis that bacterial effectors subvert the host innate immune response in intestinal epithelial cells by disrupting specific transcriptional responses to infection. As an outcome of this study, we will have determined the mechanism of a novel family of bacterial effectors that target a critical host transcription factor and will have clarified how enteric pathogens selectively modulate innate immunity. These studies will have significant positive impact on the design of new strategies to combat diarrheal disease. PUBLIC HEALTH RELEVANCE: E. coli is a significant source of food borne disease, often transmitted in undercooked beef products or contaminated vegetables. The investigator is characterizing an E. coli protein that is likely to contribute to diarrheal disease by disrupting the ability of the host immune system to respond to infection. The results from these studies are likely to suggest new therapeutic strategies for diarrheal pathogens.